The synthesis of progesterone by the corpus luteum is required for the establishment of human pregnancy. LH and hCG stimulate steroidogenesis through cAMP-dependent pathways that increase expression of the steroidogenic acute regulatory protein (StAR), a phosphoprotein that mediates the rate-limiting step in steroidogenesis. The aims of the proposed research address major gaps in our knowledge of the in vivo control of luteal progesterone production. Three specific aims are proposed: 1) To determine the patterns of StAR mRNA expression, protein levels, and phosphorylation state in the human corpus luteum throughout the luteal phase. The hypotheses to be tested are: that there is a direct relationship between progesterone production and StAR mRNA and protein levels; that specific phosphorylated forms of StAR are correlated with progesterone production; that StAR is differentially regulated in small and large luteal cells; and that diminished StAR expression and/or changes in phosphorylation state are tightly linked to functional luteolysis in contrast to the expression of steroidogenic enzymes. 2) To determine the in vivo effects of hCG on StAR mRNA expression, protein levels and phosphorylation state in corpora lutea of different ages. The hypotheses to be tested are: that hCG rapidly increases StAR mRNA and protein and affects the phosphorylation state when administered in vivo: that the stimulatory effects of hCG are greatest in the mid- and late luteal phases; and that StAR expression is differentially regulated by hCG in small and large luteal cells. 3) To determine the effects of GnRH antagonist-induced LH withdrawal on StAR expression and phosphorylation state. The hypotheses to be tested are: that withdrawal of LH in the mid-luteal phase causes a rapid decline in StAR mRNA and protein levels and a change in phosphorylation state; that changes in StAR expression precede changes in downstream steroidogenic enzyme expression and are more closely associated with falling progesterone levels; that there are differential responses of small and large luteal cells to LH withdrawal; and that administration of hCG can reverse the effects of GnRH antagonist treatment. Corpora lutea will be removed from women undergoing gynecologic surgery for benign conditions. In some cases women will be treated with hCG or a GnRH antagonist prior to surgery. Levels of StAR and steroidogenic enzyme mRNAs and proteins will be quantitated. StAR phosphorylation patterns will be analyzed by two-dimensional Western blotting. Small and large luteal cells will be isolated for analysis of StAR and steroidogenic enzyme expression. The results of these studies will provide a framework for understanding the role of StAR in the control of human luteal steroidogenesis.